Detection of enzyme activity is useful in the analysis of a biological or chemical sample, such as whole organisms, cells or cell extracts, biological fluids, or chemical mixtures. For example, information about metabolism, disease state, the identity of microorganisms, the success of a genetic manipulation, or the quantity of toxins, can be gained from evaluating the activity of certain enzymes. Furthermore enzyme conjugates are often used as sensitive bioanalytical tools for detection of analytes.
Enzyme activity is often detected through the use of a synthetic substrate. The endogenous substrates of an enzyme are used in designing synthetic substrates. Several glycosidase enzymes are known to target specific glycosides (R--O--Gly) to yield the corresponding carbohydrate and an organic alcohol or phenol (R--OH). Phosphatase enzymes catalyze the conversion of certain phosphate monoesters (R--O--P(O)(OH).sub.2) to inorganic phosphate (P.sub.i) and an organic alcohol (R--OH). Similarly, organic alcohols or phenols result when sulfatase enzymes liberate inorganic sulfate from some sulfate monoesters (R--O--SO.sub.3 H) or when guanidinobenzoatase enzymes hydrolyze aryl esters of p-guanidinobenzoic acid (R--O--(C.dbd.O)--C.sub.6 H.sub.4 --NH--(C.dbd.NH)--NH.sub.2). Carboxylic acid esters (R--O--(C.dbd.O)--R') are hydrolyzed by esterase enzymes to alcohols and acids. Cytochrome enzymes oxidize aryl alkyl ethers to give the phenol and an aldehyde or acid.
Most phosphatase and sulfatase enzymes are nonselective for the structure of the alcohol. Two types of phosphatase enzymes have been identified, however, that have different optimal pH for their enzymatic activity (pH optima about 10 and about 5 respectively). The aryl sulfatase enzyme most closely resembles the acid phosphatase in pH optimum and substrate turnover. Guanidinobenzoatase is a cell surface protease characteristic of several human tumor cell lines, which is not detectable in normal human cell strains. Esterases have structural requirements that range from those that hydrolyze esters of the lower carboxylic acids (usually &lt; about 4 carbons) to the "lipase" enzymes that optimally hydrolyze esters of the longer carboxylic acids (usually &gt; about 8 carbons). There are several cytochrome enzymes (isoenzymes) that differ in their ability to metabolize aryl ethers depending on the source of the enzyme. Table 1 lists some commonly investigated enzymes and their target groups.
TABLE 1 ______________________________________ REPRESENTATIVE ENZYMES E.C. NO. ENZYME TARGET GROUP ______________________________________ 3.2.1.20 .alpha.-Glucosidase .alpha.-D-Glucose 3.2.1.21 .beta.-Glucosidase .beta.-D-Glucose 3.2.1.22 .alpha.-Galactosidase .alpha.-D-Galactose 3.2.1.23 .beta.-Galactosidase .beta.-D-Galactose 3.2.1.24 .alpha.-Mannosidase .alpha.-D-Mannose 3.2.1.25 .beta.-Mannosidase .beta.-D-Mannose 3.2.1.30 N-Acetyl-.beta.-glucosaminidase .beta.-D-N-Acetyl-Glucos- amine 3.2.1.31 .beta.-Glucuronidase .beta.-D-Glucuronic Acid 3.2.1.38 .beta.-D-Fucosidase .beta.-D-Fucose 3.2.1.51 .alpha.-L-Fucosidase .alpha.-L-Fucose 3.2.1.-- .beta.-L-Fucosidase .beta.-L-Fucose 3.2.1.76 L-Iduronidase .alpha.-L-Iduronic Acid 3.2.1.4 Cellulase .beta.-D-Cellobiose 3.2.1.-- .alpha.-Arabinopyranosidase .alpha.-L-Arabinopyranose 3.2.1.37 .beta.-Xylosidase .beta.-D-Xylose 3.2.1.18 .alpha.-N-Acetyl-neuraminidase .alpha.-D-N-Acetyl-neuraminic acid (Sialic acid) 3.1.1-- guanidinobenzoatase aryl esters of p-guanidino- benzoic acid 3.1.3.1 alkaline phosphatase aryl or alkyl phosphate monoesters 3.1.3.2 acid phosphatase aryl or alkyl phosphate monoesters 3.1.6.1 aryl sulfatase aryl sulfate monoesters 3.3.3.41 4-nitrophenyl phosphatase aryl phosphates ______________________________________
The synthetic substrates for many enzymes, including those in Table 1 as well as many esterases and cytochrome enzymes, are consistently based on the same organic alcohol or phenolic precursors, differing only by the nature of the leaving group (e.g. phosphate, sulfate, guanidinobenzoate, carboxylic acid, carbohydrate, or alkyl alcohol). The synthetic substrate should not inhibit the enzymatic reaction so that the enzyme can produce enough product so that it can be detected (enzyme amplification of the detection product). Most synthetic substrates have been designed so that the presence of the enzyme (or enzyme conjugate) results in a detectable phenolic product, e.g. formation of a soluble colored or fluorescent product or formation of a precipitate.
Common substrates that yield soluble chromogenic (but nonfluorescent) products include phosphate or sulfate monoesters or glycosides of o-nitrophenol, p-nitrophenol, thymolphthalein and phenolphthalein. Fluorogenic substrates derived from such phenols as various 7-hydroxycoumarins, 3-O-methylfluorescein, 8-hydroxypyrene-1,3,6-trisulfonic acid, flavones or various derivatives of .alpha.-or .beta.-naphthols typically yield soluble fluorescent products. Although assays based on fluorescent products are generally preferred because of their greater sensitivity, they are deficient in a number of properties for analytical measurement of enzyme activity in vivo and in vitro.
None of the reported fluorogenic substrates that yield soluble products are optimally detected below a pH of about 6. With many substrates it is necessary to adjust the pH of the dye product to above 10 to obtain the maximum fluorescence efficiency. Assays that require such a change in pH or the addition of other development reagents are not readily adapted for highly automated analytical procedures. In addition, soluble reaction products, whether fluorescent or colored, readily diffuse away from the site of activity, especially in in vivo applications.
Certain substrates for phosphatase, sulfatase and some glycosidase enzymes are known to yield colored precipitates that are not fluorescent. The best known of these are 5-bromo-4-chloro-3-indolyl phosphate (BCIP) [Leary, et al., PROC. NATL. ACAD. SCI. 80, 4045 (1983)], 5-bromo-4-chloro-3-indolyl galactoside (X-Gal), several other "X-glycosides" that are similar to X-gal and the corresponding 5-bromo-4-chloroindolyl sulfate [Wolf, et al., LAB. INVEST. 15, 1132 (1966)]. Following enzymatic hydrolysis, the colorless 3-hydroxyindole intermediates are converted to insoluble indigoid dyes by oxidation with a second reagent or more slowly by molecular oxygen.
Menton, et al., PROC. SOC. EXP. BIOL. MED. 51, 82 (1944), introduced a two step technique in which certain phenolic products, liberated by hydrolytic enzymes, are subsequently coupled to a diazonium salt. The technique yields chromophoric, but nonfluorescent, diazo dye products. Burstone, ENZYME CHEMISTRY AND ITS APPLICATIONS IN THE STUDY OF NEOPLASM, pg. 160 (Academic Press 1962) introduced simplified simultaneous and post-coupling azo dye techniques using naphthol-AS-phosphates and sulfates, as the enzyme substrates.
A modification of the two step technique Ziomek, et al., HISTOCHEM. CYTOCHEM. 38 (3), 437 (1990), reportedly yields a red fluorescent azo dye precipitate that is useful for histochemical demonstration of phosphatase activity. The coupling reaction of the diazo color-forming reagent must be accomplished at an alkaline pH. While this pH may be adequate for histochemical detection of alkaline phosphatase activity, it does not permit continuous detection of the activity of acid phosphatase and aryl sulfatase enzymes and is suboptimal for detection of .beta.-galactosidase (pH optimum 7.2), since these enzymes all have extremely low activity in alkaline medium. Furthermore, the diazo coupling reaction is not specific for the phenols formed by the enzymatic reaction. Therefore the presence of intrinsic phenolic contaminants in the test solution or the biological fluid can yield false positive signals. All of the above methods suffer from weak and somewhat nonspecific fluorescent staining of enzyme activity.
Enzyme-amplification techniques are used in histochemistry and cytochemistry to localize specific antigens by microscopy. Success of this technique depends on an efficient site-specific deposit of enzymatic products that contrast well with the underlying cellular structures. Colored precipitate formed by hydrolysis of known chromophoric precipitating substrates such as X-gal can be well visualized at discrete loci in cells or tissues using light microscopy, if the sample has appreciable quantities of the target molecules. It has been reported that the chromophoric precipitating substrate for alkaline phosphatase, when coupled with a digoxigenin-labeled probe and anti-digoxigenin conjugated with alkaline phosphatase, can stain nerve growth factor mRNA at a higher sensitivity and resolution than a standard isotope label method [Springer et al., HISTOCHEM. AND CYTOCHEM. 39, 231 (1991)]. However, the enzymatic products from the chromophoric substrates are not sufficient to form a visible precipitate that contrasts well with cellular structures when a single molecule of the analyte must be detected, because the chromophoric signal is insufficient for detection. The fluorescent precipitate of this invention, in contrast, provides a more easily detectable signal in smaller amounts.
In recent years, numerous nonradioactive approaches have been developed and refined for in situ hybridization [Hopman et al., MOLECULAR NEUROANATOMY, pp 43, Elsevier Science Publishers (1988)]. All of these nonradioactive techniques are generally able to detect specific mRNA in situ without difficulty. In contrast, the nonradioactive methods for detecting a specific gene which exists in few or even single copies in a cell's genome using biotinylated probes, require oligonucleotides that contain several thousand bases in order to allow for sufficient incorporation of the biotin (or other) label. In practical terms, any probe shorter than about 2,000 bases will not result in visible signals sufficient to detect few or single copies in the cell genome utilizing either the colored precipitates or fluorescence microscopy. The need for a probe of such long length severely limits the ease and flexibility of the probe design because preparation involves such time-consuming techniques. Because of their stronger accumulated signal, the substrates of this invention can be used with shorter oligonucleotide probes.
The substrates of this invention also differ significantly from substrates previously described in that most known fluorogenic substrates yield products that are appreciably fluorescent only in the solution phase, whereas the preferred substrates from this invention are virtually nonfluorescent except in the solid phase. In addition they yield insoluble, highly fluorescent products without requiring addition of a color-developing and precipitating reagent. Furthermore, the subject substrates are specific for a particular enzymatic activity, and are optimally reactive at or below physiological pH. As a result of these characteristics, the substrates of this invention can detect the activity of a wide variety of enzymes and enzyme-related analytes, in living cells, in extracts of living cells, in biological fluids, in biopsy samples, in vivo and in vitro, without requiring any preprocessing of the samples by concentration, centrifugation, or filtration and without addition of secondary reagents.
Some of the fluorescent dyes used to prepare the subject substrates are already known, e.g. U.S. Pat. No. 3,169,129 2-Ortho-hydroxy-phenyl-4-(3H)-quinazolinones to Rodgers, et al. (1965) (quinazolinones); Hein, et al., The Use of Polyphosphoric Acid in the Synthesis of 2-Aryl- and 2-Alkyl-substituted Benzimidazoles, Benzoxazoles and Benzothiazoles, J. AM. CHEM. SOC. 79, 427 (1957) (benzimidazoles, benzoxazoles and benzothiazoles); and Naumann & Langhals, A Simple Synthesis of Dihydroxybipyridyls, SYNTHESIS 279 (Apr. 1990) (dihydroxybipyridyls). It has been recognized that several of the dyes have very low solubility, particularly in water, and that the compounds are fluorescent in the solid state. The large Stokes shift characteristic of some compounds in this class of dyes has also been described. There have been several studies of the fluorescence mechanism of this class of compounds which has been related to a high degree of photostability. Catalan, et al., Photoinduced Intramolecular Proton Transfer as the Mechanism of Ultraviolet Stabilizers: A Reappraisal, J. AM. CHEM. SOC. 112, 747 (1990); Sinha & Dogra, Ground State and Excited State Prototropic Reactions in 2-(o-Hydroxyphenyl)benzimidazole, CHEM. PHYSICS 102, 337 (1986); Orlando, et al., Red- and Near-infrared-luminescent Benzazole Derivatives, CHEM. COMM. 23, 1551 (1971); and Williams & Heller, Intramolecular Proton Transfer Reactions in Excited Fluorescent Compounds, J. PHYS. CHEM. 74, 4473 (1970). None of the references, however, indicate the use of these dyes as fluorogenic substrates.
Orlando, et al., supra at 1552, citing Williams & Heller supra noted that replacement of an o-hydroxyphenyl group by an o-methoxyphenyl group results in nonfluorescent benzazoles. An alkoxy group was the only blocking group described in the reference, however, and there was no indication that blocking groups could be selected to monitor the presence or activity of enzymes.